(Not applicable)
This invention relates to cell transfection using adenoviral vectors, especially replication-competent adenoviruses, and methods of their use. More Specifically, it relates to cell-specific replication of adenovirus vectors in cells expressing the androgen receptor, particularly, prostate carcinoma cells, through use of a probasin transcriptional regulatory element.
There are three significant diseases of the prostate: benign prostate hyperplasia (BPH), prostate cancer, and prostatitis. The cost of treating these three diseases is immense. The annual treatment of prostate diseases in the U.S. required 4.4 million physician visits, 836,000 hospitalizations, and cost over $3 billion in 1985. Approximately one out of every four males above the age of 55 suffers from a prostate disease of some form or another. Prostate cancer is the fastest growing cause of cancer in men, with approximately 244,000 new cases diagnosed and about 44,000 deaths reported for 1995 in the United States. Due to the aging U.S. population, the incidence of BPH and prostate cancer is likely to increase.
BPH causes urinary obstruction resulting in urinary incontinence. It occurs in almost 80% of men by the age of 80. Unregulated dihydrotestosterone is believed to cause hyperplastic prostate growth. Pharmacotherapy for the treatment of BPH is currently aimed at relaxing prostate smooth muscle (alpha blockade) and decreasing prostate volume (androgen suppression). Phase III clinical trials are underway to evaluate selective alpha, blockers, antiandrogens, and 5-alpha reductase inhibitors for the treatment of BPH. The most promising of these is finasteride, which has shown an ability to cause regression of the hyperplastic prostate gland in a majority of patients. Mocellini et. al. (1993) Prostate 22:291.
BPH is treated surgically with a transurethral resection of the prostate (TURP). This procedure is very common: 500,000 TURPs are performed in the U.S. each year and 25% of men will require surgery at some time in their lives to alleviate urinary obstruction. This makes BPH the second most common cause of surgery in males. The TURP procedure requires several days of hospitalization as well as the surgery itself. The average medical reimbursement cost of a TURP in 1987 dollars was $8,000; in 1993 dollars this is $14,000. Unfortunately, a side-effect of the TURP is the elimination of the ejaculatory ducts as well as the nerve bundles of the penis, resulting in impotence in 90% of patients. A TURP is prefaced by an outpatient biopsy procedure to determine if the enlargement of the prostate is benign or cancerous, which also adds to the cost. Hypertrophy may also be treated by transurethral insertion of a tubular stent or expandable dilation catheter to maintain the patency of the urethral lumen. U.S. Pat. No. 4,893,623, issued Jan. 16, 1990, to Rosenbluth et al.; and U.S. Pat. No. 5,527,336, issued Jun. 18, 1996, to Rosenbluth et al.
An alternative therapy for prostate diseases involves radiation therapy. A catheter has been developed which squeezes prostate tissue during microwave irradiation; this increases the therapeutic temperature to which the prostate tissue more distal to the microwave antennae can be heated without excessively heating nearby non-prostate tissue. U.S. Pat. No. 5,007,437, issued Apr. 16, 1991, to Sterzer et al. A combination of a radiating energy device integrated with a urinary drainage Foley type catheter has also been developed. U.S. Pat. No. 5,344,435, issued Sep. 6, 1994, to Turner et al.
Prostate cancer is now the most frequently diagnosed cancer in men. Prostate cancer is latent; many men carry prostate cancer cells without overt signs of disease. Autopsies of individuals dying of other causes show prostate cancer cells in 30% of men at age 50; by age 80, the prevalence is 60%. Further, prostate cancer can take up to 10 years to kill the patient after initial diagnosis. Prostate cancer is newly diagnosed in slightly over 100,000 men in the U.S. each year, of which over 40,000 will die of the disease. There is also high morbidity. Cancer metastasis to bone (late stage) is common and often associated with uncontrollable pain. Metastasis also occurs to lymph nodes (early stage).
The disease progresses from a well-defined mass within the prostate, to a breakdown and invasion of the lateral margins of the prostate, to metastasis to regional lymph nodes, to metastasis to the bone marrow. The aggressiveness of prostate tumors varies widely. Some tumors are relatively aggressive, doubling every six months, whereas other are extremely slow-growing, doubling once every five years. As a consequence of the slow growth rate, few cancer cells are actively dividing at any one time. As a result, prostate cancer is generally resistant to radiation and chemotherapy, although both therapeutic modalities are widely used. Surgery is the mainstay of treatment but it too is largely ineffective and also removes the ejaculatory ducts, resulting in impotence.
Unfortunately, in 80% of cases, diagnosis of prostate cancer is established when the disease has already metastasized to the bones. Of special interest is the observation that prostate cancers frequently grow more rapidly in sites of metastasis than within the prostate itself, the site of the primary cancer.
At this stage there is no effective cytotoxic chemotherapy for prostate cancer. Current therapeutic techniques include the use of chemical forms of medical castration by shutting down androgen production in the testes, or directly blocking androgen production in the prostate. For the treatment of prostate cancer oral estrogens and luteinizing releasing hormone analogs are used as well as surgical removal of glands that produce androgens (orchiectomy or adrenalectomy). However, estrogens are no longer recommended because of serious, even lethal, cardiovascular complications. Luteinizing hormone releasing hormone (LHRH) analogs are used instead. However, hormonal therapy invariably fails with time with the development of hormone-resistant tumor cells. It is not known whether these cells develop as a mutation of the original hormone sensitive cells, or a separate class of cells. Furthermore, since 20% of patients fail to respond to hormonal therapy, it is believed that hormone-resistant cells are present at the onset of therapy.
Estramustine, a steroidal nitrogen mustard derivative, was originally thought to be suitable for targeted drug delivery through conjugation of estrogen to toxic nitrogen mustard. Clinical trials, however, have been disappointing when survival is used as an endpoint. Finasteride, a 4-aza steroid (Proscar(copyright) from Merck and Co.), inhibits the enzyme responsible for the intracellular conversion of testosterone to dihydrotestosterone, the most potent androgen in the prostate. Casodex(copyright) is thought to inhibit cellular uptake of testosterone by blocking androgen receptors in the nucleus. However, almost all advanced cancer prostate cells fail to respond to androgen deprivation. Indolocarbazole derivatives such as K-252a have also recently been developed to treat prostate diseases. U.S. Pat. No. 5,516,771, issued May 14, 1996, to Dionne.
None of these techniques for treating prostate diseases has been universally successful. Following localized therapy, up to 40% of patients with advanced disease, and a large proportion of all patients, eventually develop metastatic disease. Treatment for advanced disease initially involving hormonal manipulations and palliative radiotherapy have demonstrated symptomatic relief, but not long-term disease-free survival. The use of cytotoxic agents in the management of hormone-resistant advanced prostate cancer remains poorly defined. A few single agents have become xe2x80x9cstandard therapyxe2x80x9d, although demonstration of their efficacy, by contemporary standards, is lacking. Combination chemotherapy is frequently employed, although its contribution to overall patient management is largely unsubstantiated, especially when critical assessment of efficacy parameters are used. Newer approaches using chemohormonal therapy and hormonal priming therapies have failed. High-dose chemotherapy with transplant regimens are not well-tolerated in an elderly population, to which most victims of prostate cancer belong. A growth factor inhibitor, suramin, has shown promising initial results. However, no therapy to date has been demonstrated to improve overall survival in patients with advanced hormone refractory prostate cancer. U.S. Pat. No. 5,569,667, issued Oct. 29, 1996, to Grove et al.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of selectivity; that is, the ability to inhibit the multiplication of tumor cells, while leaving unaffected the function of normal cells. Thus, the therapeutic ratio, or ratio of tumor cell killing to normal cell killing of traditional tumor chemotherapy, is only 1.5:1. Thus, more effective treatment methods and pharmaceutical compositions for therapy and prophylaxis of prostatic hyperplasia and neoplasia are needed.
Of particular interest is development of more specific, targeted forms of therapy for prostate diseases. In contrast to conventional cancer therapies, which result in relatively non-specific and often serious toxicity or impotence, more specific treatment modalities attempt to inhibit or kill malignant cells selectively while leaving healthy cells intact.
One possible treatment approach for prostate diseases is gene therapy, whereby a gene of interest is introduced into the malignant cell. Boulikas (1997) Anticancer Res. 17:1471-1505. The gene of interest may encode a protein which converts into a toxic substance upon treatment with another compound, or an enzyme that converts a prodrug to an active drug. For example, introduction of the herpes simplex gene encoding thymidine kinase (HSV-tk) renders cells conditionally sensitive to ganciclovir (GCV). Zjilstra et al. (1989) Nature 342: 435; Mansour et al. (1988) Nature 336: 348; Johnson et al. (1989) Science 245: 1234; Adair et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4574; and Capecchi (1989) Science 244: 1288. Alternatively, the gene of interest may encode a compound that is directly toxic, such as diphtheria toxin (DT). For these treatments to be rendered specific to prostate cells, the gene of interest can be under control of a transcriptional regulatory element that is specifically (i.e. preferentially) increases transcription of an operably linked polynucleotide in the prostate cells. Cell- or tissue-specific expression can be achieved by using cell-specific enhancers and/or promoters. See generally, Huber et al. (1995) Adv. Drug Delivery Rev. 17:279-292.
A variety of viral and non-viral (e.g., liposomes) vehicles, or vectors, have been developed to transfer these genes. Of the viruses, retroviruses, herpes virus, adeno-associated virus, Sindbis virus, poxvirus and adenoviruses have been proposed for use in gene transfer, with retrovirus vectors or adenovirus vectors being the focus of much current research. Verma and Somia (1997) Nature 389:239-242. Adenoviruses are among the most easily produced and purified, whereas retroviruses are unstable, difficult to produce and to purify, and may integrate into the host genome, raising the possibility of dangerous mutations. Moreover, adenovirus has the advantage of effecting high efficiency of transduction and does not require cell proliferation for efficient cell transduction. For general background references regarding adenovirus and development of adenoviral vector systems, see Graham et al. (1973) Virology 52:456-467; Takiff et al. (1981) Lancet 11:832-834; Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020; Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
When used as gene transfer vehicles, adenovirus vectors are often designed to be replication-defective and are thus deliberately engineered to fail to replicate in the target cells of interest. In these vehicles, the early adenovirus gene products E1A and/or E1B are deleted and provided in trans by the packaging cell line 293. Graham et al. (1987) J. Gen. Virol 36:59-72; Graham (1977) J. Genetic Virology 68:937-940. The gene to be transduced is commonly inserted into adenovirus in the deleted E1A and/or E1B region of the virus genome. Bett et al. (1994). Replication-defective adenovirus vectors as vehicles for efficient transduction of genes have been described by, inter alia, Stratford-Perricaudet (1990) Human Gene Therapy 1:241-256; Rosenfeld (1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat. Gent. 1:372-378; Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal Le Salle et al. (1993) Science 259:988-990; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem. 269:23872-23875; and Bett et al. (1994).
The virtually exclusive focus in the development of adenoviral vectors for gene therapy is use of adenovirus merely as a vehicle for introducing the gene of interest, not as an effector in itself. Replication of adenovirus has been viewed as an undesirable result, largely due to the host immune response. In the treatment of cancer by replication-defective adenoviruses, the host immune response limits the duration of repeat doses at two levels. First, the capsid proteins of the adenovirus delivery vehicle itself are immunogenic. Second, viral late genes are frequently expressed in transduced cells, eliciting cellular immunity. Thus, the ability to repeatedly administer cytokines, tumor suppressor genes, ribozymes, suicide genes, or genes which convert a prodrug to an active drug has been limited by the immunogenicity of both the gene transfer vehicle and the viral gene products of the transfer vehicle as well as the transient nature of gene expression. There is a need for vector constructs that are capable of eliminating essentially all cancerous cells in a minimum number of administrations before specific immunological response against the vector prevents further treatment.
A completely separate and unrelated area of research pertains to the description of tissue-specific transcriptional regulatory proteins.
Rat Probasin (PB) Gene
The rat probasin (PB) gene encodes a nuclear and secreted protein, probasin, that is only expressed in the dorsolateral prostate. Dodd et al. (1983) J. Biol. Chem. 258:10731-10737; Matusik et al. (1986) Biochem. Cell. Biol. 64: 601-607; and Sweetland et al. (1988) Mol. Cell. Biochem. 84: 3-15. The dorsolateral lobes of the murine prostate are considered the most homologous to the peripheral zone of the human prostate, where approximately 68% of human prostate cancers are thought to originate. Immunohistochemistry with polyclonal and monoclonal antibodies has shown dual cellular localization of PB within the cytoplasm and nucleus of epithelial cells of the prostate. The expression of this gene is mediated by both zinc and testosterone (T), or a derivative thereof, via the androgen receptor (AR). T, the dominant testicular androgen, diffuses passively into the cell and either binds directly to the AR, or undergoes enzymatic reduction to 5xcex1-dihydrotestosterone (DHT), or aromatization to estrogens. Once T or DHT binds to the AR, the protein undergoes conformational changes, chaperone proteins such as heat shock proteins dissociate from the receptor, and the activated receptor can then bind DNA. Johnson et al. (1988) in Steroid Receptors and Disease (Sheridan, ed.), pp. 207-228, Dekker, New York; and Chan et al. (1989) in Pediatric Endocrinology (Collu et al., eds.), pp. 81-124, Raven Press, New York.
The androgen-activated AR binds to specific DNA enhancer sequences called androgen-responsive elements (AREs or ARE sites). Once anchored to an ARE, the AR is able to regulate transcriptional activity in either a positive or negative fashion. Lindzey et al. (1994) Vitamins and Hormones 49: 383-432. The 5xe2x80x2 TRE (transcriptional response element) region of PB gene contains two ARE sites required for androgen regulation. Rennie et al. (1993) Mol. Endocrinol. 7:23-36; International Application PCT/CA93/00319, published as WO 94/03594, Feb. 17, 1994, to Matusik.
The AR belongs to a nuclear receptor superfamily whose members are believed to function primarily as transcription factors that regulate gene activity through binding to specific DNA sequences, hormone-responsive elements. Carson-Jurica et al. (1990) Endocr. Rev. 11: 201-220. This family includes the other steroid hormone receptors as well as the thyroid hormone, the retinoic acid and the vitamin D3 receptors. The progesterone and glucocorticoid receptor are structurally most closely related to the AR. Tilley et al. (1989) Proc. Natl. Acad. Sci. USA 86: 327-331; Zhou et al. (1994) Recent Prog. Horm. Res. 49: 249-274; and Lindzey et al. (1994) Vit. Horm. 49: 383-432.
Recently, the cDNAs encoding the human and rat AR have been cloned. Chang et al. (1988) Proc. Natl. Acad Sci. USA 85: 7211-7215; Lubahn et al. (1988) Mol. Endocrinol. 2:1265-1275; and Trapman et al. (1988) Biochem. Biophys. Res. Commun. 153: 241-248. The rat and human AR mRNAs show a high degree of sequence similarity in the coding regions and the 5xe2x80x2 UTRs.
The AR gene itself is a target of androgenic regulation. This modulation may constitute an important level of control modulating physiological effects of testosterone. Androgen promotes up- and down-regulation of AR mRNA in a tissue- and possible stage-specific fashion. Nastiuk et al. (1994) Endocrin. 134: 640-649; Shan et al. (1995) Endocrin. 136: 3856-3862; and Prins et al. (1995) Biol. Reprod. 53: 609-619. In the testis, AR protein is expressed in Sertoli cells, Leydig cells and peritubular cells, but not in the developing germ cells. Grootegoed et al. (1977) Mol. Cell. Endocrinol. 9: 159-157; and Buzek et al. (1988) Biol. Reprod. 39: 39-49. Hormones such as follicle-stimulating hormone (FSH) and testosterone affect the production of AR. Verhoeven et al. (1988) Endocrinology 122: 1541-1550; and Blok et al. (1989) Mol. Cell. Endocrinol. 63: 267-271; Quarmby et al. (1990) Mol. Endocrinol. 4:22-28.
Up- and down-regulation of AR mRNA can be reproduced in different cell lines transfected with an AR cDNA. Burnstein et al. (1995); Mol. Cell. Endocrinol. 115:177-186 and Dai et al. (1996) Steroids 61:531-539. In both COS-1 and LNCaP cells expressing an AR cDNA, androgen promotes down-regulation of AR mRNA. Burnstein et al. (1995). The prostate cancer cells lines PC3 and DU145 do not express an endogenous AR, but when these cells are transfected with AR cDNA, the gene demonstrates androgenic up-regulation. Dai et al. (1996). Both up- and down-regulation of AR mRNA in cells expressing the AR cDNA are due to sequences within the AR cDNA. The heterologous cytomegalovirus (CMV) promoter that drives the expression of the AR cDNA is not itself responsible for androgenic regulation of AR cDNA expression. Burnstein et al. (1995); and Dai et al. (1996). Therefore, androgen-mediated differential regulation of AR cDNA expression is conferred by the AR cDNA in a cell line-specific manner. Burnstein et al. (1995); Dai et al. (1996).
The molecular mechanism of AR mRNA autoregulation is complex, with both transcriptional and post-transcriptional mechanisms implicated in this process. Prins et al. (1995) Biol. Reprod. 53: 609-619; Wolf et al. (1993) Mol. Endocrin. 7: 924-936; and Blok et al. (1992) Mol. Cell. Endocrin. 88: 153-164. The 5xe2x80x2 region of the AR gene does not appear to contain AREs. Blok et al. (1992) Mol. Cell. Endocrin. 88: 153-164. The mechanism of androgen-mediated up-regulation of AR mRNA in PC3 cells (prostate cancer cell line) expressing a transfected human AR (hAR) cDNA has been studied. An androgen-responsive region within the AR coding sequence is bound by AR and contains two distinct AREs that act synergistically to mediate AR mRNA up-regulation. Dai et al. (1996) Mol. Endocrin. 10:1582-1594.
Prostate diseases are generally recalcitrant to treatment by standard therapies. Thus, it is critical to develop new therapeutic approaches for this disease. The present invention addresses this need by providing adenoviral vectors specific for replication in AR-producing cells.
All publications cited herein are hereby incorporated by reference in their entirety.
In one embodiment, the invention provides an adenovirus vector comprising an adenovirus gene under transcriptional control of a probasin transcriptional response element (PB-TRE). The PB-TRE is capable of mediating gene expression specific to cells which allow a PB-TRE to function, such as cells expressing the androgen receptor, e.g. prostate cells. The PB-TRE can comprise a promoter and/or enhancer from a probasin gene, provided that the PB-TRE is capable of mediating gene expression specific to cells expressing the androgen receptor. In one embodiment, a PB-TRE comprises a promoter from a probasin gene. In one embodiment, a PB-TRE comprises an enhancer from a probasin gene. In one embodiment, a PB-TRE comprises a promoter from a probasin gene and an enhancer from a probasin gene. In one embodiment, the PB-TRE is transcriptionally active in cells which allow a PB-TRE to function. such as cells expressing the androgen receptor (AR).
In certain embodiments, a PB-TRE comprises the nucleotide sequence of SEQ ID NO:1. In certain embodiments, a PB-TRE comprises a portion of SEQ ID NO:1 capable of mediating cell-specific transcription in AR-producing cells such as prostate cells. In another embodiment, a PB-TRE comprises the sequence from about xe2x88x92286 to about +28 relative to the transcriptional start site of a probasin gene (nucleotides about 141 to about 454 of SEQ ID NO:1). In another embodiment, a PB-TRE comprises the sequence from about xe2x88x92426 to about +28 relative to the transcriptional start site of a probasin gene (nucleotide about 1 to about 454 of SEQ ID NO:1). In another embodiment, a PB-TRE comprises the sequence to about xe2x88x92236 to about xe2x88x92223 and/or the sequence to about xe2x88x92140 to about xe2x88x92117 (nucleotides about 191 to about 204 and/or about 286 to about 310, respectively, of SEQ ID NO:1), relative to the transcriptional start site of a probasin gene, combined with a probasin or non-probasin promoter. In another embodiment, a PB-TRE comprises one or two ARE sites (androgen-responsive elements) combined with a probasin or non-probasin promoter. In each embodiment, a PB-TRE is defined as a transcriptional response element or transcriptional regulatory element capable of effecting transcription in a cell, such as a prostate cell, which allows a PB-TRE to function, such as a cell expressing androgen receptor.
In certain embodiments, the adenovirus comprises a PB-TRE, which in turn comprises at least one androgen response element (ARE). In some embodiments, the ARE is ARE-1 or ARE-2 from either the probasin gene or the AR gene. In other embodiments, the adenovirus vector comprises a PB-TRE, which in turn comprises an ARE, such as ARE-2. In other embodiments, the adenovirus vector comprises a probasin transcriptional response element, which in turn comprises both ARE-1 and ARE-2.
In some embodiments, the PB-TRE is rat in origin. In some embodiments, the rat PB-TRE is capable of mediating prostate-specific gene expression in humans.
In some embodiments, the adenovirus gene under control of a PB-TRE contributes to cytotoxicity (directly or indirectly), such as a gene essential for viral replication. In one embodiment, the adenovirus gene is an early gene. In another embodiment, the early gene is E1A. In another embodiment, the early gene is E1B. In yet another embodiment, both E1A and E1B are under transcriptional control of a PB-TRE. In other embodiments, the adenovirus gene essential for replication is a late gene. In various embodiments, the additional late gene is L1, L2, L3, L4, or L5. In another embodiment, the adenovirus gene under control of a PB-TRE is the adenovirus death protein gene (ADP).
In another embodiment, the adenovirus comprising an adenovirus gene under transcriptional control of a PB-TRE further comprises at least one additional adenovirus gene under transcriptional control of at least one additional prostate-specific transcriptional regulatory element. In one embodiment, a composition comprises this adenovirus. In one embodiment, this composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the at least one additional prostate-specific transcriptional regulatory element is a second PB-TRE. In one embodiment, the at least one additional PB-TRE can have a sequence different from that of the first PB-TRE. In one embodiment, the at least one additional prostate-specific transcriptional regulatory element comprises a prostate-specific antigen (PSA) transcriptional regulatory element.
In other embodiments, the adenovirus vector can further comprise a heterologous gene or transgene, wherein said transgene is under transcriptional control of a PB-TRE. In one embodiment, the heterologous gene is a reporter gene. In one embodiment, the heterologous gene is conditionally required for cell survival. In some embodiments, the transgene is a cytotoxic gene.
In another embodiment, a method of treating prostate cancer in an individual is provided, the method comprising the step of administering to the individual an effective amount of an adenovirus vector in which an adenovirus gene is under transcriptional control of a PB-TRE. In one embodiment, the adenovirus gene is essential for viral replication. In one embodiment, the adenovirus gene is an early gene. In one embodiment, the adenovirus gene is E1A. In one embodiment, the adenovirus gene is E1B. In one embodiment, the adenovirus gene is ADP. In one embodiment, the PB-TRE comprises an enhancer from a probasin gene. In one embodiment, the PB-TRE comprises a promoter from a probasin gene. In one embodiment, the PB-TRE comprises a promoter from a probasin gene and an enhancer from a probasin gene. In one embodiment, the adenovirus further comprises an additional adenovirus gene under transcriptional control of at least one additional prostate-specific transcriptional regulatory element. In one embodiment, the second prostate-specific transcriptional regulatory element comprises a prostate-specific antigen (PSA) transcriptional regulatory element. In one embodiment, the additional adenovirus gene is essential for viral replication. In one embodiment, the additional adenovirus gene is an early gene. In one embodiment, the additional adenovirus gene is E1A. In one embodiment, the additional adenovirus early gene is E1B. In one embodiment, the additional adenovirus gene is a late gene. In various embodiments, the late gene can be L1, L2, L3, L4, or L5. In one embodiment, the additional adenovirus gene is ADP.
In another aspect, the invention provides a host cell transformed with any adenovirus vector(s) described herein.
In another aspect, the invention provides a composition comprising an adenovirus comprising an adenovirus gene under transcriptional control of a PB-TRE. In one embodiment, the composition further comprises a pharmaceutically, acceptable excipient.
In another aspect, the invention provides kits which contain an adenoviral. vector(s) described herein.
Another embodiment of the invention is an adenovirus which replicates preferentially in mammalian cells expressing AR.
In another aspect, a method is provided for propagating an adenovirus specific for cells which allow a PB-TRE to function; such as cells expressing androgen receptor, said method comprising combining any adenovirus vector(s) described herein with cells which allow a PB-TRE to function, such as cells expressing AR, whereby said adenovirus is propagated.
In another aspect, a method for modifying the genotype of a target cell is provided, the method comprising contacting a cell which allows a PB-TRE to function, such as a cell expressing androgen receptor, with any adenovirus described herein, wherein the adenovirus enters the cell.
In another aspect, methods are provided for detecting cells expressing probasin in a biological sample, comprising contacting cells of a biological sample with an adenovirus vector(s) described herein, and detecting replication of the adenovirus vector, if any.
In one embodiment, a method is provided for detecting cells which allow a PB-TRE to function, such as cells expressing androgen receptor in a biological sample, the method comprising the steps of: contacting a biological sample with an adenovirus vector comprising a gene under transcriptional control of a PB-TRE, under conditions suitable for PB-TRE-mediated gene expression in cells which allow a PB-TRE to function, such as cells expressing androgen receptor; and determining if PB-TRE mediates gene expression in the biological sample, where PB-TRE-mediated gene expression is indicative of the presence of cells which allow a PB-TRE to function, such as cells expressing the androgen receptor. In one embodiment, the gene is a heterologous (non-adenovirus gene). In one embodiment, the heterologous gene is a reporter gene, and production of the product of the reporter gene is detected.
In another embodiment, a method is provided for conferring selective toxicity on a target cell, said method comprising contacting a cell which allows a PB-TRE to function, such as a cell expressing androgen receptor, with any adenovirus disclosed herein, wherein the adenovirus enters the cell.
In one embodiment, an adenovirus is provided which comprises a heterologous gene under transcriptional control of a PB-TRE. In one embodiment the heterologous gene is a reporter gene. In one embodiment, the heterologous gene is conditionally required for cell survival. In one embodiment, a method is provided for detecting cells which allow a PB-TRE to function, such as cells expressing androgen receptor in a sample comprising the steps of: contacting a biological sample with an adenovirus vector comprising a gene under transcriptional control of a PB-TRE, under conditions suitable for PB-TRE-mediated gene expression in cells which allow a PB-TRE to function, such as cells expressing androgen receptor; and determining if PB-TRE mediates gene expression in the biological sample, where PB-TRE-mediated gene expression is indicative of the presence of cells expressing the androgen receptor.